Cell transfection and co-immunoprecipitation

In order to check whether CacyBP/SIP co-precipitates with Hsp90, HEp-2 cells were transfected with an appropriate plasmid: pcDNA3.1-3xFLAG-Hsp90α, pcDNA3.1-3xFLAG-Hsp90β, p3xFLAG-CMV-10-Hsp90β(N+M) [encoding fragment covering the N-terminal (N) and middle (M) domain, residues 1–546], p3xFLAG-CMV-10-Hsp90β(M+C) [encoding fragment covering the middle (M) and C-terminal (C) domain, residues 206–724], p3xFLAG-CMV-10-Hsp90β(M) [encoding fragment covering the middle (M) domain, residues 206–546] or with a control plasmid p3xFLAG-CMV-10, using TrueFect Transfection Reagent (United Biosystems) according to the manufacturer's protocol. After 5 hrs, the medium was replaced with fresh complete medium and cells were cultured in 5% CO₂ at 37°C for the next 24 hrs. For co-immunoprecipitation (co-IP) cells were washed with ice-cold PBS, harvested and homogenized mechanically by being passed 40 times through a needle (0.45 x 13 mm) in the IP buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 3 mM MgCl₂, 0.1% (v/v) Triton X-100 supplemented with protease and phosphatase inhibitors (Roche). Cell lysate was centrifuged at 16 000 x g for 10 min at 4°C and protein concentration was measured by the Bradford’s procedure. Next, approximately 800–1000 μg of total protein from the supernatant fraction was incubated with anti-FLAG antibody coupled to agarose beads (Sigma-Aldrich) at 4°C for 3 hrs. The unbound fraction was removed, the resin was washed with IP buffer and the bound proteins were eluted from the anti-FLAG resin using 0.1 M glycine, pH 3.5. The eluted proteins were precipitated with cold acetone and analyzed by Western blot using specific antibodies. In order to examine the influence of Hsp90 inhibition on the CacyBP/SIP-Hsp90 complex formation, cells were treated with 1 μM radicicol or 1 mM novobiocin for 2 hrs and then harvested as described above.